**3.2.2.2 Effect of HRT on ethanol fermentation**

In order to estimate an optimal fermentation time under ultrasonic exposure in this study, parameters such as ethanol concentration, ethanol volumetric productivity, ethanol yield and lactose consumption were investigated.

The maximum values of ethanol concentration and lactose consumption were achieved when the HRT was 36 h. Under the HRT of 36 h in the ultrasound-assisted fermentation, the average ethanol concentration of 26.30 g L-1, ethanol yield of 0.532 g g-1 lactose and lactose consumption of 98,9% were obtained (Fig. 9–11). Using *S. cerevisiae* without ultrasound exposure gave the results as 23,60 g L-1, 0.511 g g-1, 92,4%, respectively and the differences were statistically significant (p<0.05). Shortening the HRT to 24 h allowed remaining high ethanol yield of 0.520 g g-1 with sonicated *S. cerevisiae*, but in the control fermentation unit it was as low as 0.487 g g-1 (p<0.05). When the HRT was 12 h the ethanol yields were 0.318 and 0.365 g g-1 depending on using ultrasounds device (Fig. 11). From the economic viewpoint, shortening the fermentation time (HRT) could reduce costs of industrial ethanol production. The study showed that there is no need to extend the HRT over 36 h or more, because most of the lactose was converted into ethanol during 24 h (95.6% in the ultrasound-assisted fermentation. Nikolić et al. (2010) stated that optimal fermentation time for free and immobilized *S. cerevisiae* was 38 h. Ozmihci & Kargi (2008) studied ethanol production from cheese whey powder (CWP) solution containing 50 g sugar L-1 at six different HRTs varying between 17.6 and 64.4 h by *Kluyveromyces marxianus* strains. Percent sugar utilization,

that discontinuous ultrasonic irradiation of *S. cerevisiae* was more beneficial for activating fermentation than the continuous exposure, because only a few steps in intracellular

Liu et al. (2007) investigated the changes of biological activity of aerobic activated sludge after ultrasonic irradiation. The activity of microorganisms rose sharply after ultrasonic exposure of 0.3 W cm2, 35 kHz for 10 min, and reached a peak level in 8 h after exposure (100% higher than that of the initial level immediately after exposure). Then it dropped rapidly in the next 8 h. In 24 h after ultrasonic irradiation, the enhancement effect induced by ultrasound almost disappeared, and the cells activity returned to the normal state as control cells without ultrasound stress. The authors stated that the enhancement might be due to defense response of microorganisms evoked by the mechanical stress. That reactions

Pitt & Ross (2003) used ultrasonic irradiation to increase the growth rate of bacterial cells attached to a polyethylene surface. It was found that low frequency ultrasound (70 kHz) of low intensity (<2 W cm-2) increased the growth rate of the cells compared to growth without ultrasonic waves. They stated that ultrasounds can increase the rate of transport of oxygen and nutrients to the cells and the rate of transport of waste products away from the cells,

Xie et al. (2009) studied the enhancement effect of low-intensity ultrasound (35 kHz) on anaerobic sludge activity. The experiments showed, that the optimal ultrasonic intensity and

To sum up, the optimal ultrasonic intensity and irradiation period are varied in each

In order to estimate an optimal fermentation time under ultrasonic exposure in this study, parameters such as ethanol concentration, ethanol volumetric productivity, ethanol yield

The maximum values of ethanol concentration and lactose consumption were achieved when the HRT was 36 h. Under the HRT of 36 h in the ultrasound-assisted fermentation, the average ethanol concentration of 26.30 g L-1, ethanol yield of 0.532 g g-1 lactose and lactose consumption of 98,9% were obtained (Fig. 9–11). Using *S. cerevisiae* without ultrasound exposure gave the results as 23,60 g L-1, 0.511 g g-1, 92,4%, respectively and the differences were statistically significant (p<0.05). Shortening the HRT to 24 h allowed remaining high ethanol yield of 0.520 g g-1 with sonicated *S. cerevisiae*, but in the control fermentation unit it was as low as 0.487 g g-1 (p<0.05). When the HRT was 12 h the ethanol yields were 0.318 and 0.365 g g-1 depending on using ultrasounds device (Fig. 11). From the economic viewpoint, shortening the fermentation time (HRT) could reduce costs of industrial ethanol production. The study showed that there is no need to extend the HRT over 36 h or more, because most of the lactose was converted into ethanol during 24 h (95.6% in the ultrasound-assisted fermentation. Nikolić et al. (2010) stated that optimal fermentation time for free and immobilized *S. cerevisiae* was 38 h. Ozmihci & Kargi (2008) studied ethanol production from cheese whey powder (CWP) solution containing 50 g sugar L-1 at six different HRTs varying between 17.6 and 64.4 h by *Kluyveromyces marxianus* strains. Percent sugar utilization,

biological process enhanced by ultrasound and should be find experimentally.

metabolisms are supported by ultrasound and others are not or even inhibited.

are usually observed when cells are challenged by biotic or abiotic stresses.

irradiation period were 0.2 W cm2 and 10 min, respectively.

**3.2.2.2 Effect of HRT on ethanol fermentation** 

and lactose consumption were investigated.

thus enhancing their growth.

Fig. 9. Effects of HRT and ultrasound irradiation on the ethanol concentration

Fig. 10. Effects of HRT and ultrasound irradiation on the lactose consumption by coimmobilized *S. cerevisiae*

Feasibility of Bioenergy Production from

fermentation process was recorded.

**3.2.2.3 Effect of ultrasounds on ethanol fermentation** 

Ultrafiltration Whey Permeate Using the UASB Reactors 209

In all HRTs, significant higher ethanol productions in the ultrasound-assisted fermentation process than in the control fermentation process were recorded (p<0.05). When the HRT was 12 h, the ethanol concentration without ultrasonic treatment was 9.87 g L-1 and it was significant lower by 2.85 g L-1 than the production in the process stimulated with low intensity ultrasounds (p<0.05) (Fig. 9). Lactose consumption was only 62.1%, but application of ultrasound increased it to 69.7% (p<0.05) (Fig. 10). The best results were obtained with the longest HRT of 36 h. Ethanol concentration increased to the value of 26.30 g L-1 when the culture has been sonicated, while in the fermentation process without ultrasound irradiation was only 23.60 g L-1 (p<0.05), (Fig. 9). Lactose consumption was as high as 98.9% in ultrasound-assisted fermentation unit and was significant higher by 6.5% than the consumption in the reactor without ultrasonic irradiation (p<0.05) (Fig. 10). High ethanol production and lactose consumption were observed with shortening HRT to 24 h. *S. cerevisiae* stimulated with low intensity ultrasound produced 24.85 g ethanol L-1, while the lactose consumption was 95.6% (Fig. 9–10). In the control fermentation unit there was 21.79 g L-1 and 89.5%, respectively. The differences were statistically significant (p<0.05). Under the HRT of 36 h, in the fermentation process with ultrasound irradiation the maximum ethanol yield of 0.532 g g-1 lactose was observed, whereas using biocatalyst *S. cerevisiae* without ultrasound exposure gave the result as 0.511 g g-1 (Fig. 11) (p<0.05). Shortening the HRT to 24 h allowed remaining high ethanol yield of 0.520 g g-1 with sonicated *S. cerevisiae*, but in the control fermentation process it was as low as 0.487 g g-1 (p<0.05). When the HRT

was 12 h the ethanol yield was only 0.365 and 0.318 g g-1, respectively (p<0.05).

There were only few experiments investigating the enhancing ethanol production by ultrasonic stimulation of *S. cerevisiae*. Schläfer et al. (2000) improved biological activity of *S. cerevisiae* by low energy ultrasound assisted bioreactors operated at a frequency of 25 kHz and a power input of 0.3 W L-1. The ethanol production without ultrasonic treatment varied between 3–12 g L-1, while ultrasonic stimulation increased it to 30 g L-1. The highest ethanol concentrations were obtained with a cycle regime of ultrasound exposure and a pause, because during continuous ultrasound irradiation no stimulation in the ethanol

Lanchun et al. (2003) investigated the influence of low intensity ultrasound on physiological characteristic of *S. cerevisiae*. The results of their study showed, that ultrasounds in the frequency of 24 kHz and the power efficiency of 2 W with 1 s irradiation time every 15 s and 30 min duration cycle, stimulated the material transport and improved the cell's metabolism by changing the osmotic pressure of membrane. Consequently, transfer of substance was

The positive results of the ultrasound treatment on the ethanol production by coimmobilized *S*. *cerevisiae* seemed to be a combination of different processes, including activating the yeast by improving the mass transfer rate of nutrients in the liquid, enhancing the uptake of foreign substances and the release of intracellular products in cells, improving the cell growth and degassing of CO2 (Lanchun et al. 2003; Liu et al., 2007; Liu et al. 2003b). Stimulating enzyme activity is done by increasing in the mass transfer rate of the reagents to the active site (Liu et al., 2007). Ultrasounds irradiation can cause thermal and mechanical stress to biological materials (Liu et al., 2003b). High energy ultrasonic waves break the cells

speeded up, enzyme synthesis was driven up and enzyme activity was enhanced.

Fig. 11. Effect of HRT and ultrasound irradiation on the ethanol yield

effluent ethanol concentration and ethanol yield increased with increasing HRT from 17.6 to 50 h. Further increasing in HRT to 64.4 h resulted in decrease of the analyzed parameters. Moreover, the time for fermentation decreased at higher initial substrate concentration (Guimarães et al., 2008a; Nikolić et al., 2010; Ozmihci & Kargi, 2008). According to Guimarães et al. (2008a) the fermentations with 50-150 g lactose L-1 reached completion in about the same time of 27 h but the maximum ethanol concentration increased linearly with increasing initial lactose concentration from 6.5 g ethanol L-1 with 20 g lactose L-1 to 57 g L-1 with 200 g L-1. They also stated that increasing lactose concentration led to incomplete fermentation and impair the fermentation due to nutrient limitation.

Interestingly, the volumetric productivities of ethanol decreased at longer HRT (Table 3). Maximum productivity of ethanol of 1.060 g L-1 h-1 was observed under the HRT of 12 h when the culture has been sonicated and 0.908 g L-1 h-1 under the HRT of 24 h in the fermentation process without ultrasound irradiation (p<0.05). The volumetric ethanol productivity in the ultrasound-assisted fermentation obtained in this work was higher than that reported for batch or fed-batch fermentations with *S. cerevisiae* strains: 0.3 g L-1 h-1 (Rubio-Texeira et al., 1998), 0.46 g L-1 h-1 (Guimarães et al., 2008b), 0.14 – 0.6 g L-1 h-1 (Ramakrishnan & Hartley, 1993), 1 g L-1 h-1 (Compagno et al., 1995). Ozmihci & Kargi (2007) using *Kluyveromyces marxianus* to ferment concentrated cheese whey powder solution obtained higher volumetric ethanol productivity over 2 g L-1 h-1, but after 120 h fermentation.


Table 3. Effects of HRT on the ethanol volumetric productivity

Ultrasonic irradiation Without ultrasonic irradiation

12 24 36

HRT (h)

effluent ethanol concentration and ethanol yield increased with increasing HRT from 17.6 to 50 h. Further increasing in HRT to 64.4 h resulted in decrease of the analyzed parameters. Moreover, the time for fermentation decreased at higher initial substrate concentration (Guimarães et al., 2008a; Nikolić et al., 2010; Ozmihci & Kargi, 2008). According to Guimarães et al. (2008a) the fermentations with 50-150 g lactose L-1 reached completion in about the same time of 27 h but the maximum ethanol concentration increased linearly with increasing initial lactose concentration from 6.5 g ethanol L-1 with 20 g lactose L-1 to 57 g L-1 with 200 g L-1. They also stated that increasing lactose concentration led to incomplete

Interestingly, the volumetric productivities of ethanol decreased at longer HRT (Table 3). Maximum productivity of ethanol of 1.060 g L-1 h-1 was observed under the HRT of 12 h when the culture has been sonicated and 0.908 g L-1 h-1 under the HRT of 24 h in the fermentation process without ultrasound irradiation (p<0.05). The volumetric ethanol productivity in the ultrasound-assisted fermentation obtained in this work was higher than that reported for batch or fed-batch fermentations with *S. cerevisiae* strains: 0.3 g L-1 h-1 (Rubio-Texeira et al., 1998), 0.46 g L-1 h-1 (Guimarães et al., 2008b), 0.14 – 0.6 g L-1 h-1 (Ramakrishnan & Hartley, 1993), 1 g L-1 h-1 (Compagno et al., 1995). Ozmihci & Kargi (2007) using *Kluyveromyces marxianus* to ferment concentrated cheese whey powder solution obtained higher volumetric ethanol productivity over 2 g L-1 h-1, but after 120 h

> Ethanol volumetric productivity in the control fermentation system (g L-1 h-1)

Fig. 11. Effect of HRT and ultrasound irradiation on the ethanol yield

fermentation and impair the fermentation due to nutrient limitation.

Ethanol volumetric productivity in the ultrasound-assisted fermentation system (g L-1 h-1)

Table 3. Effects of HRT on the ethanol volumetric productivity

12 h 1.060 0.822 24 h 1.035 0.908 36 h 0.730 0.655

0.25

fermentation.

HRT

0.3

0.35

0.4

Ethanol yield (g g-1)

0.45

0.5

0.55
